Structural and functional insight into the regulation of kinesin-1 by microtubule associated protein MAP7

The microtubule (MT)-associated protein, MAP7 is a required cofactor for kinesin-1 driven transport of intracellular cargoes. Using cryo-electron microscopy and single-molecule imaging, we investigated how MAP7 binds MTs and facilitates kinesin-1 motility. The MT-binding domain (MTBD) of MAP7 bound MTs as an extended α-helix between the protofilament ridge and the site of lateral contact. Unexpectedly, the MTBD partially overlapped with kinesin-1’s binding site and inhibited kinesin-1 motility. However, by tethering kinesin-1 to the MT, the projection domain of MAP7 prevented dissociation of the motor and facilitated its binding to available neighboring sites. The inhibitory effect of the MTBD dominated as MTs became saturated with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive binding to MTs.

sites. The inhibitory effect of the MTBD dominated as MTs became saturated with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive binding to MTs.

One Sentence Summary:
MAP7 binds to microtubules as an extended α-helix and activates kinesin-1 motility despite the overlap between their tubulin binding sites.
Kinesin and dynein are microtubule-associated molecular motors that are differentially regulated to deliver intracellular cargos to their destinations (1,2). Intracellular cargos are sorted by structural MAPs that decorate the MT surface (1). Distinct cellular localizations of MAPs correlate with their regulatory roles in intracellular traffic (3). Overexpression of tau disrupts kinesin-1 (kinesin hereafter) transport of synaptic vesicles in axons (4,5), while the knockdown of tau rescues defects in axonal transport in Alzheimer's disease models (6). Unlike tau, MAP7 is a required cofactor for kinesin-driven transport in cells (7)(8)(9). The MAP7 projection domain binds to kinesin's coiled-coil stalk in vitro (9,10), recruits kinesin to MTs, and activates its motility (9,11). Transient interactions with the MAP7 projection domain may enable kinesin to hop from one MAP to another, increasing its apparent run length by disfavoring detachment from the MT (9).
To understand how MAP7 regulates kinesin, we determined the cryo-electron microscopy (cryo-EM) structure of MTs decorated with full-length (FL) MAP7 ( Fig. 1A-B, fig. S1A, Tables S1-2, and movie S1). The reconstruction revealed a 53 residue-long α-helix that runs parallel to the MT axis about the length of a tubulin dimer ( Fig. 1A-C, fig. S1B). Unlike MAP2, MAP4, and tau, which bind along the outer ridges of the protofilaments (12)(13)(14), MAP7 ran halfway between the outer ridge and the site of lateral contacts. The co-structure of MAP7's MT binding domain (MTBD; residues 60-170) and FL tau on the MT ( fig. S2) illustrated their distinct MT footprints and confirmed that the helical segment corresponds to the MAP7's MTBD. We identified a single MAP7 sequence register that corresponds to a well conserved segment of the MTBD (residues 87-139; fig. S3A-C) via Rosetta modeling (15), and validated this registry by determining the structure of MTs decorated with a shorter MAP7 construct (residues 83-134; fig. S3D). Because MAP7 MTBD could potentially form a helix longer than the length of a tubulin heterodimer ( fig. S2A), we cannot exclude the possibility of a larger footprint of MAP7 on the MT ((13), fig. S4 and Methods).
The α-helical density for MAP7 is not uniform (Fig. 1B). Segment I (residues 113-139), which is the best-resolved region (Fig. 1B), makes extensive interactions with tubulin ( Fig.  1D): Q113 and E117 of MAP7 are within hydrogen-bonding distance of N197 and S155 of β-tubulin, respectively; R114 and K127 of MAP7 engage in electrostatic interactions with E159 and D414 of β-tubulin, respectively. Y108 of β-tubulin inserts into a hydrophobic pocket formed by R120, R121, and V124 of MAP7. We also identified potential hydrogen bonds between R128, R131, and K136 of MAP7 and the mainchain oxygens of E411 and G410 of β-tubulin and V159 of α-tubulin, respectively. Segment III (residues 87-99) interacts with α-tubulin. Segment II (residues 100-113) faces a cavity at the intra-tubulin dimer and has the weakest density in our map (Fig. 1B). All-atom molecular dynamics (MD) simulations verified these pairwise interactions, and additional potential interactions between MAP7 and tubulin ( fig. S5, movie S2). Segment II fluctuated more than other segments in simulations because it only makes transient contacts with tubulin ( Fig. 1E-F,  fig. S5).
We next determined how MAP7 affects the motility of kinesin and the dynein-dynactin-BicDR1 complex (DDR, dynein hereafter) (16) (Fig. 2A). FL MAP7 uniformly decorated MTs with a dissociation constant of 111 ± 12 nM (±s.e.) under physiological salt (150 mM KAc, Fig. 2B-C, fig. S6). As previously reported (9,11), the addition of 50 nM MAP7 rescued FL kinesin from autoinhibition (17) and substantially increased its run frequency and length ( fig. S7). MAP7 also enhanced the motility of constitutively-active kinesin (K560, residues 1-560) ( Fig. 2D-E, movie S3) (9,11). Unlike run length and frequency, kinesin velocity decreases even at low MAP7 concentrations (Fig. 2E), which could be because of pausing at MTBD obstacles or binding to the projection domain. Although dynein has been reported not to be inhibited by 5 nM MAP7 (10), we found that it was inhibited by MAP7 with half-max inhibition constant of 10 ± 3 nM ( Fig. 2D-E). MAP7 decoration of MTs also switched the direction of an assembly that links FL kinesin to dynein ( Fig. 2F) (18). While 80% of kinesin-dynein assemblies were minus-end-directed on undecorated MTs, 93 % moved towards the plus-end in 10 nM MAP7 ( Unexpectedly, kinesin run frequency decreased when MAP7 concentration was increased further (100-1,000 nM, Fig. 2D-E, movie S3). We reasoned that the nonlinear relationship between MAP7 decoration and kinesin motility could arise when the motor is subjected to simultaneous activation and inhibition that dominate at different concentrations ( Fig. 2E) (19). To determine the source of these opposing inputs, we disrupted the kinesin-MAP7 interaction by truncating either the MAP7-binding domain of kinesin (K490, residues 1-490) or the kinesin-binding domain of MAP7 (MAP7-N and MAP7-MTBD, Fig. 3A). In all cases, MAP7 inhibited kinesin (Fig. 3B, fig. S8 and movies S5-6) as strongly as it inhibited dynein (Fig. 2E). Thus, MTBD inhibits while the projection domain activates kinesin (10). MTBD may inhibit kinesin by competing for the same tubulin binding site, because superimposing the MT-bound structure of kinesin (20) onto our model reveals an apparent clash between kinesin and segment II of MAP7 ( fig. S9A). Alternatively, the flexible segment II may accommodate kinesin binding by shifting away from the intradimer interface, as proposed for DCX and MAP4 (14,21). To distinguish between these possibilities, we determined the structure of MTs incubated with rigor kinesin (K350 E236A ) and MAP7 (Fig. 3C). Because distinct binding sites of MAP7 and kinesin can be artifactually averaged during reconstruction ( fig. S9B), we performed focused 3D classification around the putative shared binding site of MAP7/kinesin ( fig. S10, see Methods). The classification resulted in two distinct maps of the binding site, one occupied by MAP7 only, and the other occupied by kinesin only (Fig. 3D). Thus, kinesin and MAP7 cannot simultaneously bind to the same tubulin dimer.
Consistent with cryo-EM, MTBD was unable to bind MTs pre-decorated with K350 E236A (Fig. 3E). However, FL MAP7 or a construct that lacks the MTBD (ΔMTBD, fig. S11A-C) still bound to kinesin-decorated MTs to a significant extent, presumably via the P123 domain (residues 175-316) (8,9). MT binding of ΔMTBD was nearly abolished upon cleaving the flexible tails of tubulin (Fig. 3E, fig. S11D), explaining why P123 was invisible in our structure. To verify that kinesin is inhibited at high MAP7 decoration owing to binding site overlap, we also replaced the MT-binding regions of MAP7 with that of tau, which also overlaps with kinesin's binding site and inhibits kinesin motility ( fig. S12) (13,22). Similar to FL MAP7, binding of this chimeric MAP to the MT resulted in biphasic regulation of kinesin motility (Fig. 3F-G).
To reveal how kinesin walks along MAP7-decorated MTs despite competing for the same binding site, we fluorescently labeled the motor domain of K560 and tracked kinesin stepping with nanometer precision under limiting ATP conditions (Fig. 4A). On undecorated MTs, kinesin took 16-nm steps in the forward direction (23), whereas steps in the sideways and backward directions were rare (9% and 3%, respectively; Fig. 4A-B, fig. S13A) (24). At 36 nM MAP7, we observed a modest increase in the probability of sideways and backward stepping ( Fig. 4A-B). At nearly saturating MAP7 concentrations, we observed 16-64 nm displacements in our trajectories, as well as increased stepping in sideways (35%) and backward (26%) directions ( Fig. 4A-B). Because kinesin cannot take such large steps on its own (Fig. 4A-B), these large displacements likely represent transient detachment and reattachment of the motor to the MT.
In the absence of the projection domain, kinesin was stuck on MTs decorated with 36 nM MTBD and dissociated from MTs at increased ATP concentrations ( fig. S13B-C). Furthermore, the projection domain needed to be tethered to the MTBD to supersede kinesin inhibition, because the addition of the C-terminal half of MAP7 (MAP7-C) was unable to stimulate kinesin motility on MTs decorated with the N-terminal half of MAP7 (MAP7-N, fig. S14). Thus, the projection domain is essential for kinesin to bypass MTBD obstacles.
Based on our results and previous reports (9,11), we propose a model for kinesin stepping on MAP7 decorated MTs. MAP7 recruits kinesin-1 to the MT and activates subsequent motility (9,11). MT binding of MAP7 also obstructs kinesin stepping along the protofilament, which results in kinesin dissociation from the MT. However, the MAP7 projection domain tethers kinesin to the MT surface, allowing it to rebind the MT at nearby sites not blocked by its MTBD. This "tethered diffusion" of kinesin appears as large forward, sideways, or backward displacements in our trajectories. When the MT surface is nearly saturated with MAP7, the frequency and length of kinesin runs are reduced because of the scarcity of empty tubulin sites to which the motor can rebind after it dissociates (Fig.  4C). Unlike kinesin-1, MAP7 inhibits dynein motility because its MTBD overlaps with the dynein binding site ( fig. S15) but its projection domain does not tether dynein to MTs. This biphasic regulation mechanism may enable precise control of kinesin-1-driven transport by varying MAP7 density on cellular MTs. While MAP7 is required for kinesin-1 driven processes in many cell types (10,25,26), dense MAP7 localization at branch junctions in rat neurons slows down and pauses organelles at these sites (8). This mechanism may reroute cargos to their destinations by facilitating the detachment of kinesin from its MT track and its switching to neighboring MTs at cellular junctions (8).

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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.     The MAP7 projection domain rescues kinesin from autoinhibition and tethers the motor to the MT. When kinesin encounters an MTBD obstacle, it dissociates from the MT, remains tethered to MAP7, and rebinds to the available tubulin site on another protofilament. Kinesin is inhibited at high MAP7 concentrations due to the scarcity of available binding sites.